title: Protease inhibitor from insect silk-activities of derivatives expressed in vitro and in transgenic potato.
abstract: Several recombinant derivatives of serine protease inhibitor called silk protease inhibitor 2 (SPI2), which is a silk component in Galleria mellonella (Lepidoptera, Insecta), were prepared in the expression vector Pichia pastoris. Both the native and the recombinant protease inhibitors were highly active against subtilisin and proteinase K. The synthetic SPI2 gene with Ala codon in the P1 position was fused with mGFP-5 to facilitate detection of the transgene and its protein product. A construct of the fusion gene with plant regulatory elements (promoter 35S and terminator OCS) was inserted into the binary vector pRD400. The final construct was introduced into Agrobacterium tumefaciens that was then used for genetic transformation of the potato variety Velox. The transgene expression was monitored with the aid of ELISA employing polyclonal antibody against natural SPI2. In vitro tests showed increased resistance to the late blight Phytophthora infestans in several transformed lines. No effect was seen on the growth, mortality, life span or reproduction of Spodoptera littoralis (Lepidoptera, Insecta) caterpillars, while feeding on transformed potato plants expressing the fusion protein, indicating that the transformed potatoes may be harmless to non-target organisms.
authors.affiliation: Institute of Entomology, Biology Centre ASCR, Branišovská 31, 370 05, České Budějovice, Czech Republic. kodrik@entu.cas.cz
authors.forename: Dalibor
authors.initials: D
authors.lastname: Kodrík
authors.forename: Barbara
authors.initials: B
authors.lastname: Kludkiewicz
authors.forename: Oldřich
authors.initials: O
authors.lastname: Navrátil
authors.forename: Oxana
authors.initials: O
authors.lastname: Skoková Habuštová
authors.forename: Vendulka
authors.initials: V
authors.lastname: Horáčková
authors.forename: Zdeňka
authors.initials: Z
authors.lastname: Svobodová
authors.forename: Konstantin S
authors.initials: KS
authors.lastname: Vinokurov
authors.forename: František
authors.initials: F
authors.lastname: Sehnal
mesh_terms: D000818:Animals; D060467:Disease Resistance / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology; D015870:Gene Expression; D005818:Genetic Engineering / Q000009:adverse effects / Q000379:methods*; D019476:Insect Proteins / Q000235:genetics*; D007915:Lepidoptera / Q000235:genetics*; D055750:Phytophthora infestans / Q000502:physiology; D010843:Pichia / Q000235:genetics; D010935:Plant Diseases / Q000469:parasitology; D030821:Plants, Genetically Modified; D011198:Solanum tuberosum / Q000166:cytology / Q000235:genetics* / Q000276:immunology / Q000469:parasitology; D018411:Spodoptera; D020860:Subtilisin / Q000037:antagonists & inhibitors / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D019476:Insect Proteins; C425550:silk proteinase inhibitor 2, Galleria mellonella; D020860:Subtilisin